I Helicobacter pylori genes involved in immune-system evasion In a previous collaboration with Drs. Ding Jin and Yan Zhou, NCI, we asked whether the spoT gene of H. pylori plays a role in environmental stresses and intracellular survival. The SpoT gene encodes a bifunctional enzyme with both (p)ppGpp synthetase and hydrolase activities. We made the following observations: 1) SpoT mutants failed to produce (p)ppGpp when shifted from rich to minimal media lacking carbon sources and phosphates, 2) ATP levels increased with growth for 48 hours in all strains but the peak value for ATP in the spoT mutant was reproducibly lower than wt, 3) The spoT mutant converts from spiral form to coccoid form prematurely, 4) SpoT mutants have a longer lag time when grown microaeophilically with low CO2 (2.5% vs. 10%). Finally, the macrophage intracellular survival of spoT mutants decreased 4 to 10-fold relative to isogenic wt H. pylori strains after 24 hour. In contrast, both spoT mutant and isogenic wt strains were phagocytized by macrophage, at the same rate and equal numbers of engulf bacteria were observed after 1 hour. These results suggest that the spoT mutation reduced the intracellular survival in macrophage only. Recently we extended this study by testing in vivo the survival of both spot mutant and isogenic wt strains in mice and in a macrophage cell line, RAW Cell 264.7 (RAW Cells). Helicobacter pylori SS1 SpoT +/+ bacteria have a survival advantage in the stomachs of Mice and within Raw Cells, of 10- and 7-fold respectively. Studies have been initiated to elucidate the mechanistic role that SpoT plays in the intracellular and gastric survival of H. pylori. Our first study utilized the two-hybrid system for identifying SpoT-protein-interactions. This study identified 28 SpoT-protein interactions. We have begun analyzing a number of these protein-interactions to identify the pathways involved and understand the function of the SpoT protein. Previous research resulted in H. pylori specific antibiotic-based in vivo expression technology (IVET) systems that were used to identify in vivo induced (ivi) genes in mice and cultured macrophage. These IVET screenings identified 31 novel in vivo induced genes (ivi genes) which had not been previously reported. We are continuing to investigate these 31 novel ivi genes as possible additional virulence factors essential for colonization or pathogenesis of the stomach mucosa. In vitro and in vivo studies of these H. pylori mutant strains are continuing with the goal of identify novel ivi genes that play a role in stomach colonization. II Host mucosal and immune responses to Helicobacter pylori infection In collaboration with Dr. Griffin Rodgers research group, Molecular and Clinical Hematology Branch, NHLBI, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4-/- mice have increased intracellular killing of Staphlocococus aureus and Escherichia coli in vitro. The OLFM4-/- mice also have enhanced in vivo bacteria clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by intra-peritoneal injection. OLFM4 is found to interact with cathepsin C. We demonstrated that OLFM4 is a direct substrate of cathepsin C and inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4-/- mice is significantly higher than that in neutrophils from wild-type littermate mice. The activities of serine proteinases, neutrophil elastase, cathepsin G and proteinase 3, which require cathepsin C activity for processing and maturity, are also higher in OLFM4-/- neutrophils. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases. We have recently shown experimentally that, H. pylori infection induces a Vitamin D Immune response. A vitamin D antimicrobial activity against Mycobacterium tuberculosis was confirmed in human monocytes in 1986 and 1987. However, there are few, if any, reports indicating a vitamin D3 immune response to Helicobacter pylori (H. pylori) infection. We used microarray analysis to monitor host responses to H. pylori infection and found that the vitamin D receptor gene (VDR) was up-regulated (fold changes>5, p<0.05), which suggested that VDR may play an important role in immune response to H. pylori infection. We tested this observation in the RAW 264.7 cell line using quantitative PCR, and confirmed that VDR, CYP27B1 and Cathelicidin expressions were increased during H. pylori infection. Further, we also observed increased CYP27b1 expression, 1, 25-dihydroxyvitamin D3 (1,25D3) levels and Cathelicidin expression in resident macrophages isolated from the peritoneal cavity of C57BL/6 wild type mice. In contrast, CYP27b1 is down-regulated in resident macrophages isolated from VDR-deficient C57BL/6 VDR KO mice. We have now extended our studies to C57BL/6 wild type and C57BL/6 VDR KO mice to evaluate the role of VDR on the modulation of mucosal immune response to H. pylori infection. H. pylori colonization in the gastric mucosa of C57BL/6 VDR KO mice was significantly lower compared with wild-type littermates. Together these observations indicate that vitamin D3 exerts considerable influence on the host innate immune response against H. pylori infection via the CYP27b1 response and subsequent roles of VDR and 1,25D3 on Cathelicidin production. Our results are in agreement with previous studies showing that Vitamin D3 immune response inhibits the adaptive immune responses, which may contribute to persistence of H. pylori colonization. We have used several mouse models to investigate H. pylori infection, pathogenic mechanism(s) and mucosal-immune responses. We selected wild type (wt) C57BL/6 and knockout mice to evaluate the role of several endogenous gene products on the regulation of mucosal immune response to H. pylori infection. The knockout mice allowed us to examine the in vivo role of a number of genes (or gene products) on bacterial load per gram of stomach tissue and pathology. We monitored histological changes by pathology scoring. In collaboration with Dr. Richard DiPaolo, Saint Louis University Medical School, we developed a new technique that allows us to isolate the inflammatory cells from the stomach mucosa of mice. Using this technique, we identified quantified, and determined the functions and specificities of cells infiltrating the stomach after infection. We observed increases of CD4+ and CD8+ T cells infiltrating the stomach tissue of wt infected mice. We are also studying innate and adaptive immunity as they relate to H. pylori infection. The cellular and molecular mechanisms that initiate H. pylori adaptive immunity and T-cell response are poorly understood. Our goal is to assess the relative contribution of MyD88 and Toll-like receptors 2 and 4 in host response to H. pylori infection and to monitor T-cell responses. Ongoing, animal studies confirmed an IL-17 local immune response and a significant increase in infiltration of CD4+ and CD8+ T-cells to H. pylori infection of gastric tissue. Flow cytometric studies of stomach infiltrates indicated that CD4+ T-cells were the primary T-cell subset involved in the IL-17 immune response. Our results clearly indicate that Helicobacter pylori induces IL-17 signaling in murine models.